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Indian J Med Res 126, July 2007, pp 34-38

Antinuclear antibodies by indirect immunofluorescence : Optimum


screening dilution for diagnosis of systemic lupus erythematosus

P. Ghosh, S. Dwivedi, Sita Naik, Vikas Agarwal, Anupam Verma*, Amita Aggarwal & Ramnath Misra

Departments of Immunology & *Transfusion Medicine, Sanjay Gandhi Postgraduate Institute of Medical
Sciences, Lucknow, India

Received October 9, 2006

Background & objectives: Antinuclear antibodies (ANA) are serological hallmark of systemic lupus
erythematosus (SLE). Conventionally, the test is carried out on human epithelial cells (HEp2) by
indirect immunofluorescence (IIF) technique. Since culturing and maintaining HEp2 cells in the
laboratory are labour intensive, in-house assays have given way to kits manufactured by commercial
companies. The reference screening dilutions provided by the manufacturers are based on different
ethnic population than ours. Therefore, it becomes mandatory for every laboratory to have its own
screening dilutions for the local population that distinguishes best between healthy and diseased
state. As, there is paucity of such data, we aimed to define the optimum screening dilution that
distinguishes the patient with SLE from healthy individuals.
Methods: Sera of patients fulfilling ACR criteria for diagnosis of SLE, idiopathic inflammatory
polymyositis/dermatomyositis (PM/DM) and rheumatoid arthritis (RA), and age and sex matched
healthy individuals were tested for ANA by IIF using a commercial kit (Euroimmun, Germany) at
5 dilutions, namely 1:40, 1:80, 1:160, 1:320 and 1:640. Receiver operator characteristics (ROC)
curve were constructed to define the optimum dilution that distinguished healthy sera from the
diseased ones.
Results: Test was performed on 213 sera from 94 healthy individuals, and 43 SLE, 37 RA and 39
DM/PM patients. In healthy individuals, ANA at dilutions 1:40, 1:80, 1:160, 1:320 and 1:640 was
positive in 13.8, 4.3, 2.1, 2.1 and 0 per cent respectively, whereas in SLE it was positive in 95.3, 95.3,
65.1, 53.5 and 23.3 per cent respectively.
Interpretation & conclusion: ROC curves analysis showed that at 1:40 dilution, sera of 95.3 per cent
of SLE and 13.8 per cent of normal individuals were (ANA) positive, whereas at 1:80 dilution it was
95.3 per cent for SLE and 4.3 per cent for healthy individuals. A fluorescent intensity of 2 was
more specific for SLE. The best discrimination between healthy individuals and the SLE patients
was found at screening dilution of 1:80 and fluorescent intensity of 2 in our laboratory.

Key words Antinuclear antibodies - indirect immunofluorescence - screening dilutions - systemic lupus erythematosus

Antinuclear antibodies (ANA) are the serological immunofluorescent assay with rodent liver tissue as the
hallmark of systemic lupus erythematosus (SLE), substrate 1. With the increasing awareness among
originally described in 1957 using an physicians of autoimmune connective tissue diseases,
34
GHOSH et al : ANA SCREENING DILUTION 35

this test is frequently used by clinicians since it is the American College of Rheumatology (ACR) criteria for
mainstay of diagnosis of SLE. diagnosis of SLE6 were included in the study. Healthy
volunteers between the ages of 18 and 65 yr, and who
Since its original description, the technique has
gave no history of any illness and had no physical or
undergone considerable methodological modifications
mental disability were selected from among the staff of
and the rodent tissue substrate has been replaced with
human epithelial cell lines such as the human epithelial the hospital and members of their family. Five ml of
(HEp2) cells. This has the advantages of increased blood was collected; and serum separated within 4 h and
sensitivity, easier identification of different patterns of stored at -20C till use. Stored sera (-80C) from patients
reactivity and recognition of antibody specificities that with dermatomyositis/polymyositis (DM/PM)7 and
react with antigens associated with dividing cells2. The rheumatoid arthritis (RA)8 who fulfilled the diagnostic
culture and maintenance of cell lines in the laboratory criteria of each of these diseases were also tested. The
is labour intensive and requires considerable expertise. study was not submitted to the institutional ethics
Hence, in house assays have been replaced by committee for the approval because this was a validation
commercial kits. These kits contain HEp2 coated slides, exercise undertaken to improve the quality of the test.
antibody conjugates and reference sera. There have also However, a written informed consent was undertaken
been recent advances in the technology of coating HEp2 from all healthy volunteers and SLE patients who
cells onto glass slides that is being adopted by some participated in the study.
manufacturers and this adds to the variability in the All the kits (Euroimmun, Germany) were purchased
sensitivity of different kits. Any increase in sensitivity and tests were carried out without any knowledge or
usually compromises specificity and this is particularly obligation to the manufacturer. The study was performed
relevant in the case of ANA detection since the test is with the same batch of the kit from the manufacturer and
also positive in a large variety of connective tissue as per the instructions provided. The kit contained HEp2
diseases besides SLE, such as mixed connective tissue slides, sample diluents, antibody conjugate, control sera
disorder, primary Sjogrens syndrome, rheumatoid and wash buffer. The assay was performed according to
arthritis and in malignancies and infectious diseases3. manufacturers instruction. Briefly, 25 ml of diluted sera
ANA positivity is also seen in normal individuals, the (1:40) were layered on HEp-2 cell spots and incubated
frequency increasing with age4. for 30 min. The slide was washed by flush of phosphate
Currently all the kits being used in Indian laboratories buffer saline (PBS)Tween and then immersed in PBS
are imported. The standardization of these kits has been for 5 min. Later 20 ml of fluorescein-labeled (goat) anti-
done using normal and diseased sera of non Indian origin. human globulin was added to each spot and slide
Since both genetic and environmental factors are known incubated for another 30 min. Again the slide was washed
to influence autoantibody production5, it may be expected in PBS-Tween for 5 min as described earlier, embedded
that the prevalence in healthy individuals will be different with 10 ml of embedding medium and read under the
in our country. This is especially important in view of the fluorescent microscope (Olympus; Japan).
high infection load in our population. Hence, it is relevant Two independent observers (SN and VA) experienced
that the imported kits, which are being used for diagnostic in reading ANA slides, blinded to the diagnosis and the
tests in our population, should be validated using serum interpretation of the other observer read the slides. Prior
samples from local population with a view to define the to the actual test both the investigators had participated
screening dilutions. We therefore carried out this study in several sessions of reading ANA slides to agree on a
to define the dilution to be used for distinguishing the consensus. ANA was defined as positive at a particular
patient with SLE from normal individuals and establish dilution if it was read positive by both the observers at
the frequency of ANA positivity in well defined patients that dilution. Both positive and negative control sera were
with other connective tissue diseases. provided by the manufacturer. All sera that were positive
Material & Methods at the 1:40 dilution were tested at 1.80, 1:160, 1:320 and
1:640 dilutions. The screening dilution recommended by
The study was conducted at the department of the manufacturer was 1:100.
Immunology, Sanjay Gandhi Postgraduate Institute of
Medical Sciences, Lucknow, between July 2004 to The criterion for assigning intensity of fluorescence
December 2005. Healthy volunteers from among the staff was as follows: 4+ (very bright green), 3+ (bright green),
of the hospital and consecutive patients who fulfilled 2 + (green), 1 + (faint green).
36 INDIAN J MED RES, JULY 2007

Statistical analysis : All statistical analyses were carried


out on SPSS software (11.5 version). Interclass correlation
coefficient was used to look for inter-observer variability.
The chi-square test of significance was used to find out
any difference in ANA positivity in different age groups of
healthy controls. The receiver operator characteristic
(ROC) analysis was used to determine the cut-off level
that differentiates between healthy and diseased individuals.
While generating ROC curves larger test results were taken
to indicate more positive test and standard error of area
with distribution assumption as nonparametric and
confidence levels of 95 per cent were used. ROC analysis
was also done to assess which definition of the fluorescent
positivity (1 or 2) gives better cut-off to differentiate
healthy individuals from patients with SLE.
Results
Fig. 1. ANA positivity at different dilutions in patients with SLE,
Test for ANA was performed on 213 serum samples DM/PM and RA.
collected from 94 healthy controls (59 females, median
age 35 yr, range 18-61), 43 SLE (35 females, median ANA in systemic autoimmune disorders :The percentage
age 33 yr, range 23-47), 37 RA (26 females, median age of sera positive for ANA in systemic autoimmune
35 yr, range 25-65) and 39 DM/PM patients (27 females, diseases is shown in Fig. 1. Very few patients of RA
median age 44 yr, range 35-64). The age distribution of had ANA positivity at 1:320 or 1:640 in contrast to SLE
healthy controls (Table I) shows that the number of and DM/PM.
subjects in the 56-65 yr group was less than in other age Determining the dilution that distinguishes healthy
groups. individuals from SLE patients : The ROC curve (Fig. 2)
Variability : Inter-observer variability gave an interclass showed that at a dilution of 1:40, 95.3 per cent of SLE
coefficient of 0.938, [95% confidence interval (95% CI) patients and 13.8 per cent of healthy individuals were
0.92-0.95] at 1:40 dilution, 0.964 (95%CI, 0.953- 0.972) ANA positive. At a dilution of 1:80, 95.3 per cent of the
at 1:80 dilution and 0.905 (95%CI, 0.821- 0.95) at 1:160 SLE patients and 4.3 per cent of healthy individuals were
dilution. Thus, the inter-observer variability was low at ANA positive (Table II). This indicated that 1:80 dilution
all the three dilutions. was the best screening dilution to distinguish SLE
patients from healthy individuals.
ANA in healthy subjects : There was no difference in
ANA positivity among different age groups (Table I). ROC analysis to determine whether fluorescent intensity
Overall, ANA was positive in 13.8, 4.3, 2.1, 2.1 and 0 of 1 or 2 is a better cut-off for ANA positivity : At a
per cent of healthy controls at dilutions 1:40, 1:80, 1:160, dilution 1:80 the sensitivity of ANA for diagnosis of
1:320 and 1:640 respectively. SLE was no different between fluorescence intensity of
1 or 2 but there was better specificity at a fluorescence
intensity of 2 (95.7% vs 90.4%) as compared to
intensity of 1 (Fig. 2, Table II).
Table I. ANA positivity in sera from healthy controls in different
age groups Discussion
Age No. of Dilutions This is perhaps the first study from our country to
groups indivi- 1:40 1:80 1:160 1:320 1:640 determine the optimum screening dilution for ANA using
(yr) dual
a commercial kit. This validation has important clinical
18-25 19 2 0 0 0 0 implications as ANA is being widely used due to ease of
26-35 33 4 1 1 1 0 availability as well as increasing awareness among
36-45 19 4 2 1 0 0
46-55 18 2 0 0 0 0
medical fraternity.
56-65 5 1 1 0 0 0 Our observation of 1:80 being the best screening
Total 94 13 4 2 1 0 dilution for diagnosis of SLE is in contrast to the
GHOSH et al : ANA SCREENING DILUTION 37

(a) (b)

Fig. 2. ROC curves of ANA for diagnosis of SLE (a) taking intensity of fluorescent (IF) > 1, and (b) taking intensity of fluorescent > 2 as
positive.( ) 1:40 positive, ( ) 1:80 positive, ( ) 1:160 positive, ( ) 1:320 positive, ( ) 1:640 positive).

Table II. Sensitivity and specificity taking intensity of fluorescence (IIF)1 and 2 as positive
Sensitivity (%) Specificity (%)
Disease Dilution
IIF intensityI IIF intensity2 IIF intensityI IIF intensity2
SLE 1:40 95.3 93 86.2 89.4
1:80 95.3 95.3 90.4 95.7
1:160 88.4 65.1 93.6 97.9
1:320 69.8 53.5 95.7 97.9
1:640 53.5 23.3 100 100
DM/PM 1:40 51.3 51.3 86.2 89.4
1:80 48.7 46.2 90.4 95.7
1:160 46.2 41 93.6 97.9
1:320 38.5 30.8 95.7 97.9
1:640 23.1 17.9 100 100
RA 1:40 45.9 43.2 86.2 89.4
1:80 13.5 5.4 90.4 95.7
1:160 10.8 8.1 93.6 97.9
1:320 8.1 8.1 95.7 97.9
1:640 2.7 2.7 100 100
SLE - Systemic lupus erythematosus
DM/PM Dermatomyositis/polymyositis
RA Rheumatoid arthritis

prevalent practice of using sera at 1:40 dilution. This is individuals. The percentage of ANA positivity in our
reflected in a survey9 by the college of American study at 1:40 was lower than that reported by Tan et al
Pathologists which, found that 59.6 per cent of the where 31.7 per cent of healthy persons were ANA
laboratory use 1:40 as a cut-off, 23.1 per cent use positive. The lower percentage in our study could be
1:80, 14 per cent use 1:160 and rest use other cut- explained by difference in ethnicity and environmental
offs. Tan et al10 have shown that 1:160 was the best factors. However, our percentage was more that those
dilution which distinguished SLE patients from healthy reported from Middle East countries where it is reported
38 INDIAN J MED RES, JULY 2007

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Reprint requests: Prof. Ramnath Misra, Department of Clinical Immunology, Sanjay Gandhi Post Graduate Institute of Medical
Sciences, Lucknow 226014, India.
e-mail : rnmisra@sgpgi.ac.in